Arthropod-borne Rickettsia species are among the most virulent and lethal human pathogens and are of significant global Public Health concern. Our work over the past 3.5 years resulted in significant progress in understanding of R. typhi pathogenesis, and in particular, the role of the rickettsial secretome during host cell invasion. For this proposal, we will address fundamental questions in two understudied yet highly significant topics in rickettsial pathogen biology. First, we will determine the mechanisms by which R. typhi modulates cat flea (Ctenocephalides felis) innate immunity, microbiota and other factors to establish infection and facilitate transmission to the vertebrate host; and second, defining the functional spectrum and immunogenic potential of the R. typhi armory of secreted phospholipases that alter host membrane physiology to facilitate phagosomal escape and intracellular replication. We will implement a transdisciplinary approach (e.g., phylogenomics, bacteriology, entomology, and cellular/molecular biology) to thoroughly investigate these focal areas, as outlined by the following Specific Aims. In Aim I, we will decipher the modulatory factors R. typhi employs to colonize C. felis and facilitate transmission via 1) a robust tissue-specific, time-course expression analysis, 2) silencing IMD and Toll pathway gene expression, 3) testing the immunomodulatory propensities of isolated R. typhi peptidoglycan, and 4) characterizing the impact of the C. felis microbiome on infection. In Aim II, we will characterize the function and immunogenicity of R. typhi secretory phospholipases. We will take two approaches to characterize R. typhi phospholipases: 1) functional characterization of Pat1, Pat2 and Pld via determining their subcellular localization, lipid recognition and interactome within host cells, with downstream analyses designed to reveal the significance of these interactions in mediating bacterial access to the host cytosol; and; 2) identification of inflammasome components necessary for IL-1? secretion by R. typhi, and determining the impact of Pat1, Pat2, and Pld activities on inflammasome activation. Together these studies will significantly advance our understanding of the complex Rickettsia/vector/host relationship, shedding light on how rickettsial parasites of arthropods transition to vertebrate pathogens throughout the obligatory Rickettsia life cycle. Thus, we anticipate that our work will take us closer to generating more prudent therapeutics to combat fatal rickettsioses.